Lyme disease (LD) is the most commonly reported tick-borne illness in the United States. According to the recent Center for Disease Control and Prevention report, approximately 300,000 Lyme cases are diagnosed each year. If left untreated, the infection spreads to joints, the heart and the nervous system. In most cases, infections and associated symptoms are eliminated by antibiotic use. Following the antibiotic treatment, however, many patients have persistent or recurrent symptoms characterized by lingering fatigue, pain, or joint and muscle aches. Studies in animal models indicate that Lyme spirochetes may persist following antibiotic therapy. For those patients who do not respond antibiotics, a novel therapy based on bispecific antibodies is proposed. Bispecific T-cell engaging antibodies (BiTe) will used to redirect T cells to borrelial target cells to trigger cytotxic action by T-lymphocytes. In Phase I, single domain nanobodies to human T cell protein CD3 and Lyme bacteria therapeutic target VlsE, DbpA and CRASP-1 with high affinity and specificity will be developed. Isolated nanobodies will be humanized to reduce potential immunogenicity in humans and respective BiTe antibodies will be produced. The activity of BiTe antibodies to the targets will be characterized in in vitro immunoassays and cytotoxicity assays. In Phase II, the BiTe antibody characterization will be extended to animal models infected with Lyme bacteria to demonstrate its anti-Lyme therapeutic effects.